Project Summary/Abstract During the epithelial-mesenchymal transition (EMT), cells undergo a switch from a polarized, epithelial state to a highly motile and more aggressive mesenchymal phenotype that is associated with cancer stem cells (CSC) and metastases (1,2). Our lab has identified a regulatory mechanism by which Transforming Growth Factor-? (TGF?) induces EMT at the translational step of gene expression (3-5). An inhibitory complex consisting of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) and eukaryotic elongation factor 1a1 (eEF1a1) suppresses translational elongation through binding to the 3'UTR of several EMT-inducing genes. TGF? stimulation promotes the release of this complex by Akt2-mediated phosphorylation of hnRNP-E1 at Ser43. Among the mRNAs controlled by this mechanism is the cytokine Interleukin-like EMT Inducer (ILEI), a secreted factor belonging to the FAM3 family. The biochemical mechanism of the ILEI pathway and identification of the ILEI receptor has remained elusive. The basis of this proposal aims to investigate the mechanism of the ILEI signaling pathway and its contribution to EMT and CSC formation. In order to identify potential ILEI binding partners, we utilized yeast two-hybrid screening and observed that ILEI interacts with the 190kD leukemia inhibitory factor receptor (LIFR). LIFR is cross-reactive with several cytokine ligands and forms a complex with glycoprotein 130 (GP130) and an array of other ligand-specific co- receptors during signal transduction (9,10). In order to investigate whether ILEI signals through the LIFR/GP130 complex, we have expressed and purified a construct of ILEI that can rescue TGF?-mediated EMT in cells with ILEI silenced as well as induce activation of LIFR/GP130. Therefore, we hypothesize that ILEI-induced EMT and CSC formation is achieved through its ligand binding and activation of the LIFR/GP130 receptor complex. We will address this hypothesis through a set of specific aims. Aim 1 will validate the LIFR/GP130 receptor complex as the bona-fide ILEI receptor. We will show that ILEI interacts with LIF-R and induces cross phosphorylation of both LIFR and GP130 that further activates the Jak/STAT pathway. These results will allow for establishment of an ILEI-specific gene-signature. Aim 2 will elucidate the contribution of ILEI signaling to CSC formation, tumorigenesis and metastasis in vivo through modulation of the ILEI pathway. We will show that abrogation of ILEI signaling will subsequently block these phenotypes. Taken together, this proposal will confirm the molecular mechanism of ILEI signaling, promoting future studies into the development of targeted drug therapies against TGF?-mediated breast cancer metastasis.